Enhanced blood product antiviral process and product produced

ABSTRACT

A transfusion blood product container for the introduction of one or more blood products, such as whole blood, platelet concentrations, leukocyte concentrations, plasma, phasma derivatives, whole blood fractions, and combinations thereof, for transfusing the patient and an amount of one or more glycyrrhizic triterpenoid compounds sufficient to comprise from 0.05 to 10.0 wt/%, preferably from about 0.5 to about 3 wt/%, of the contents of the container when full of the blood product(s), sufficient to substantially inactivate viruses contained in the blood product introduced into said container is disclosed. One or more additional products are added to the glycyrrhizic triterpenoid compounds to produce a synergistic affect.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of my copending U.S. patent applicationsSer. No. 07/321,522, filed Mar. 9, 1989, now abandoned, Ser. No.07/290,161, Filed Dec. 28, 1988, now U.S. Pat. No. 4,891,222, and Ser.No. 07/276,113, filed Nov. 23, 1988, now abandoned, to which priority isclaimed.

FIELD OF THE INVENTION

This invention relates to the treatment of mammalian biological cellsand fluids with one or more of a class of compounds referred to here asglycyrrhizic compounds, exemplary of which are glycyrrhizin,glycyrrhizinic acid or glycyrrhetinic acid glycoside, and analogoustriterpenes, e.g. carbenoxolone and cicloxolone and their derivatives,to inactivate virus and to improved containers for providing suchtreatment.

BACKGROUND OF THE INVENTION

This invention relates to the treatment of whole blood to inactivate ordestroy infective viruses found in animal fluids and cells, such as thecytomegalovirus which is responsible for or aggravates serious andsometimes fatal infections, in blood transfusion recipients. The term"inactivate" as applied to the inactivation of virus in accordance withthe present invention means that the virus are rendered non-infectiousto the patient. In some instances, virus cultures may still be viablefor a period of time, but lack the ability to pathogenically infect apatient.

Cytomegalovirus (CMV) is probably the most ubiquitous of the pathogenicviruses found in animal fluids and tissues. Virtually all of the peopleliving in the developing countries become infected with CMV early inlife, and CMV infects over half the population in the developedcountries of the world. CMV may remain essentially inactive in the bodyfollowing an initial infection and may flare in to an active infectionany time, most frequently when the body's immune system is compromisedto a greater or lesser degree by disease, radiation therapy, drugtherapy, surgical trauma, etc. CMV is frequently associated with, andmay be a causative or contributing factor in, lifethreatening disease inindividuals with suppressed immune systems, and can be a principalcausative factor in pneumonia, neurological disorders, febrile illness,ocular disease and hepatitis. CMV infection is a serious limiting factorin the transplantation of organs, tissues and cells and the transfusionof blood and plasma from one individual to another. The kidneytransplant patient runs a high risk of contracting serious, and notinfrequently fatal, CMV infection from CMV introduced by the transplantorgan. Recipients of whole blood, plasma, bone marrow, cornea, cardiac,and semen run a serious risk of CMV infectious disease, the risk beingmultiplied where the immune system of the recipient is suppressed toprevent rejection of the foreign organ or cells, or whereimmunosuppression is present from natural causes.

CMV is frequently associated with Pneumoncystis carinii and may cause orcontribute to encephalitis and colitis and may be associated withKaposi's sarcoma in AIDS patients. CMV is so ubiquitous in the blood andorgans of donors who, frequently, exhibit no symptoms of infection, andits direct and contributory effects in infectious diseases is sopervasive and subtle that a CMV infection is to be presumed if anothercausative agent cannot be established.

There are no proven cures or generally effective drugs for the treatmentof CMV infections. Certain drugs, e.g. ganciclovir, has been shown tohave limited effectiveness in the treatment of certain CMV infections,e.g. CMV retinitis, but has little effect in the treatment of CMVpneumonia. Live attenuated CMV vaccine has been developed but may notprotect against infection by natural CMV, and there is a real risk thatthe attenuated CMV may reactivate during pregnancy and infect the fetus.

While a method of preventing, or even reducing the likelihood oftransmitting CMV via transfusion or transplant of organs, tissues, cellsor fluids would be of enormous benefit to medical science, the presentinvention is not limited to treatments to inhibit CMV infection and isapplicable to other classes of viruses found in animal fluids andtissues.

CMV is a member of the human herpesvirus (HV) group, which areresponsible for much of mankind's discomfort and pain. The herpesvirusesrepresent a very large, clearly defined group of viruses which areresponsible for, or involved in, cold sores, shingles, a venerealdisease, mononucleosis, eye infections, birth defects and probablyseveral cancers. Three subfamilies are of particular importance. Thealpha subfamily includes HV-1 (herpes virus simplex 1) which causes coldsores, fever blisters, eye and brain infections, HV-2 (herpes virussimplex 2) which cause genital ulceration, and HV-3 (HV varicellazoster) which causes chicken pox, shingles and brain infections. Thebeta subfamily includes HV-5, the principal member of which is CMVdiscussed above. The gamma subfamily includes HV-4 (Epstein-Barr) whichcause infectious mononucleosis and is involved in Burkitt's lymphoma andnasopharyngeal carcinoma. Additional possibly pathogenic herpes virusesno doubt exist, one type of which, HV-6, of unknown pathogenicity hasbeen identified. (Niederman, J. C. et al., The Lancet, Oct. 8, 1988,817). There is evidence that the methods of this invention are effectivein inhibiting the transmission of infections caused by many and perhapsall of the pathogenic herpes viruses found in animal fluids and tissues.

While blood bankers have instituted rigid criteria for exclusion ofpotential donors in high risk categories, this is not a satisfactorysolution to the most significant threat to face the health carecommunity in many decades. Institution of human immunodeficiency virus(HIV) testing has blood products safer, but the complete elimination ofHIV contaminated blood and blood products has not been possible usingpresent knowledge and technology. The ELISA test, for example, missesapproximately 1 in 200 (0.5%) HIV infected donors, and there is nocertain method for excluding donor carriers of hepatitis and otherinfectious viruses found in animal fluids and tissues. Increasingefforts are exerted to improve the safety of the blood supply such asretrovirus screening using surrogate markers, screening for HIV andother retroviruses with attention to population surveillance for neweragents, cleaner methods of extracting specific blood components bymonoclonal antibody techniques and DNA methodologies, development ofrecombinant DNA products which by-pass the need for plasma derivedclotting factors for administration to hemophiliacs. Careful screeningof donors, followed by antibody testing, reduces the risk of AIDS andother virus-contaminated blood, but such methods are not foolproof. Suchmethods require testing supplies and trained technicians which are notavailable and are too expensive for use in such places as central Africaand other third-world countries where AIDS infects up to onethird of thepopulation. A simpler and less costly method of handling blood isrequired in such areas of the world.

A photodynamic method has also been evaluated as a means of eradicatingviral contaminants (Matthews, J. L. et al., Transfusion, 28,1 1988) buthas not been proved to be generally effective and safe. While FactorVIII products may be rendered non-infectious by heat orsolvent-detergent methods, no methods are known to guarantee the safetyof whole blood or cellular components or plasma. For the whole bloodrecipient, however, the only reasonably reliable safety procedures areprograms allowing for self donation prior to elective surgery by thedonor and the use of blood from designated donors, but such programs areincredibly difficult logistically. In spite of heroic efforts to meetthe challenge of virus contaminated blood supply, an imperative needcontinues to exist for a method for treating whole blood for use intransfusion.

It is apparent from the foregoing discussion that a method of killing orinactivating pathogenic viruses in organs, tissues, cell and fluidsintended for transfusion or transplantation would be an enormous advancein medicine. It is to this major national and worldwide health carechallenge that the present invention is directed.

My copending U.S. patent application Ser. No. 07/290,161, Filed Dec. 28,1988, describes and claims a method for inactivating virus in bloodsamples using glycyrrhizic triterpenoid compounds.

The invention described and claimed herein is an improvement thereof andis based upon the discovery of unique synergistic improvement resultswhen glycerol, even trace amounts of glycerol, are combined in the bloodor blood product sample with glycyrrhizic triterpenoid compounds.

Further, the invention described and claimed herein is an improvementthereof and is further based upon the discovery of unique synergisticimprovement results when even trace amounts of ethylene diaminetetraacetic acid or salts thereof (EDTA), are combined in the blood orblood product sample with glycyrrhizic triterpenoid compounds.

SUMMARY OF THE INVENTION

The invention is embodied, inter alia in an improved transfusion bloodcontainer for the introduction of one or more blood products, such aswhole blood, platelet concentrations, leukocyte concentrations, plasma,plasma derivatives, whole blood fractions, and combinations thereof, fortransfusing the patient, the invention comprising an improvedtransfusion blood product container and an amount of one or moreglycyrrhizic triterpenoid compounds sufficient to comprise from 0.05 to10.0 wt/%, preferably from about 0.5 to about 3 wt/% and a viralinactivation enhancer, e.g. glycerol or EDTA, or both, of the contentsof the improved container when full of the blood product(s), sufficientto substantially inactivate viruses contained in the blood productintroduced into said improved container. There is a striking synergismbetween the glycerol and the glycyrrhizic triterpenoid compound(s)rendering the combination surprisingly effective in inactivatingsusceptible viruses which may be in the blood product. Even such traceamounts of glycerol as would be ineffective as viral inactivatorconstitutes in blood products enhance the viral inactivation value ofGTPD many fold.

This invention relates to improved containers for collecting andtreating whole blood with GTPD compounds, e.g. glycyrrhizic acid, itsanalogues such as carbenoxolone and cicloxolone, analogues thereof andthe salts, esters and other derivatives thereof, and a viralinactivation enhancer, e.g. glycerol or EDTA, or both, and to wholeblood for transfusion containing such compounds which is free of CMVvirus capable of infecting the recipient of such blood. Inactivation ofother viruses found in animal fluids and tissues also results.

This invention is embodied inter alia in improved methods of treatmentof mammalian cells and fluids with one or more of a class of compoundsreferred to here as glycyrrhizic compounds, exemplary of which areglycyrrhizin, glycyrrhizinic acid or glycyrrhetinic acid glycoside, andanalogous triterpenes, e.g. carbenoxolone and cicloxolone and theirderivatives, and a viral inactivation enhancer, e.g. glycerol or EDTA,or both, to inactivate virus found in animal fluids and tissues, such ascytomegalovirus, bovine diarrhea virus, human immunodeficiency virus andhepatitis viruses.

This invention involves improved methods and improved containers forcollecting and treating transfusion blood products, i.e. blood productsderived from transfusion quality whole blood, including whole blood asone such product, with GTPD compounds, e.g. glycyrrhizic acid, itsanalogues such as carbenoxolone and cicloxolone, analogues thereof andthe salts, esters and other derivatives thereof and a viral inactivationenhancer, e.g. glycerol or EDTA, or both,, and to whole blood fortransfusion containing such compounds which is free of CMV virus capableof infecting the recipient of such blood. Inactivation of other virusesfound in animal fluids and tissues also results. At least one of theretroviridae is susceptible to the treatment of this invention,according to presently available data. The most notorious of theretroviridae, HIV-1, the only virus thus far identified as inducing AIDSin humans, is inactivated and/or killed using the improved methods andcompositions of this invention. Other retroviridae are considered to besusceptible to the present invention, and treatment to preventtransmission of retrovirus-infected organs, tissues, cells and fluids iswithin the scope of this invention. The treatment of such organs,tissues, cells and fluids to prevent the transmission ofhepandnaviridae-related infections, e.g. hepatitis, is also within thescope of this invention, but data regarding the actual effect oftreatments on hepatitis infectivity is so difficult to obtain thatreliable data proving the efficacy of the present invention in hepatitisinfection inhibition are extremely difficult to obtain.

This invention is embodied in the process of collecting blood from adonor comprising withdrawing the blood from the donor and introducingthe blood into a whole blood container which contains an effectiveamount of a GTPD compound consisting essentially of glycyrrhizin,glycyrrhetinic acid, carbenoxolone, cicloxolone and analogues andderivatives thereof, or mixtures thereof, and one or more viralinactivation enhancers, e.g. glycerol or EDTA, or both, for inactivatingHIV and other viruses found in animal fluids and tissues and preventingcoagulation of the collected blood.

This invention is also embodied in commercial blood containers whichcontain an amount of a GTPD compound which consists essentially ofglycyrrhizin, glycyrrhetinic acid, carbenoxolone, cicloxolone analoguesand derivatives thereof, or mixtures thereof in combination with atleast one viral inactivation enhancer, e.g. glycerol or EDTA, or both,in an amount effective to inactivate HIV and/or other viruses found inanimal fluids and tissues.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Glycyrrhizic acid,20B-carboxy-11-oxo-30-norolean-12-en-3B-yl-2-OB-D-glucopyranuronsyl-.alpha.-D-glucopyranosiduronicacid, commonly known as glycyrrhizin, glycyrrhizinic acid orglycyrrhetinic acid glycoside (also referred to as biosone, enoxolone,and glycyrrhetin) an extract from Glycyrrhiza, better known as licorice,an extract of the dried rhizome and roots of Glycyrrhiza glabra, is atriterpene and is exemplary of the triterpenes to which this inventionrelates. Analogous triterpenes to which this invention relates includecarbenoxolone and cicloxolone. This invention thus relates toglycyrrhizic acid and analogues thereof, in the form of acids, salts,esters and other derivatives. Many such derivatives are known, such as,for example, glycyrrhetinyl stearate; monopotassium glycyrrhetin;potassium glycyrrhetinate; 11-deoxoglycyrrhetinic acid hydrogen maleatesodium salt; α-D-glucopyranosiduronic acid monoarginine glycyrrhizinate;18α-Glycyrrhizic acid monosodium salt; 18-α-Glycyrrhizic acidmonopotassium salt; disodium 18-α-glycyrrhizate; glycyrrhizinic acidmono(triethanolamine) salt; trisodium glycyrrhizinate; sodiumglycyrrhizate; ammonium glycyrrhizinate; sodium carbenoxolone(biogastrone; glycyrrhetinic acid hydrogen succinate disodium salt); andacetylglycyrrhetic acid (glycyrrhetinyl acetate). Glycyrrhizin and thevirucidal analogues and derivatives thereof are referred to forconvenience herein as glycyrrhizic triterpenoids abbreviated GTPD.

Ring-substituted derivatives of GTPD compounds are contemplated and areincluded in this invention. Halogen ring substituents, such as, forexample, fluoro- and chloro- substituents, sulfate and other activeand/or inactivating substituents to the ring structure of GTPD compoundsare specifically included in this invention, without excluding otherring-substituted derivatives of GTPD compounds.

In addition to its use as a flavoring agent, licorice has long been acommon folk medicine for the treatment of sore throats. While not widelyknown, various extracts of and preparations derived from licorice, e.g.glycyrrhizin and its derivatives, principally the salts of glycyrrhizicacid, have also been used to a limited degree for many years as anorally administered medication for the treatment of peptic ulcers(Chandler, R. F., Can. Pharm. J., V118, No. 9, 1985), and oraladministration of glycyrrhizin contemporaneously with saponinantiinflammatory agents has been reported to inhibit saponin andsaponigen hemolysis (Segal, R. et al., Biochem. Pharmacol. 26, 7 1977).

GTPDs have been evaluated extensively in vitro, and have beenadministered orally, intramuscularly and intravenously. No significanttoxicity from limited, short term administration of glycyrrhizin hasbeen reported. Adverse reactions have been reported in certain instancesof prolonged oral ingestion and a slight relapse after rapiddiscontinuation of intravenous administration of Stronger Neo-MinaphagenC (SNMC) solution, glycyrrhizin (0.2%), cysteine (0.1%) and glycine (2%)was attributed to the steroid ring in glycyrrhizin (Fujisawa K. et al.,Asian Med. J. (Japan), 23, 10 1980). Dosages of SNMC as high as 60ml/day (˜12 mg/dy of glycyrrhizin) have been reported (Iwamura K.,Therapiewoche (W. Germany) 30, 34 1980).

Inactivation of viruses, in vitro, under certain conditions, has beenreported (see, e.g., Pompei R., Exprientia (Switzerland) 36/3 1980).Such anti-viral activity as GTPD compounds sometimes exhibit has beenattributed to reverse transcriptase-inhibitory activity (Nakashima, H.et al., Jpn. J. Cancer. Res. 78, 8 1987) and to enhancement ofinterferon-gamma production (Shinada, M. et al., Proc. Soc. Exp. Biol.181, 2 1986), but the exact mechanism of the anti-viral function has notbeen confirmed.

Dargan, D. J., and Subak-Sharpe, J. H., (J. Gen. Virol., 1985-1986)reported antiviral action of carbenoxolone and cicloxolone on herpessimplex virus. Their dose-response experiments showed cicloxolone sodiumor carbenoxolone sodium interfered with the HSV replication cycle andreduced the infectious virus yield by 10,000- to 100,000-fold,cicloxolone being the more potent anti-herpes agent, but no consistenteffect on HSV DNA synthesis was identified. Some inhibition of cellularDNA synthesis was observed, but this was relatively slight.

Csonka, G. W. and Tyrrell, D. A. (Br. J. Vener. Dis. 1984, 60 (3) p178)undertook a double blind clinical study to compare the efficacy ofcarbenoxolone and cicloxolone creams with placebo in initial andrecurrent herpes genitalis and reported significant differences in thetime to disappearance of pain and the healing of lesions usingcicloxolone, but carbenoxolone showed insignificant beneficial effect.

GTPDs have also been evaluated therapeutically as anti-viral agents inthe chemotherapy of acquired immune deficiency syndrome (AIDS) (Ito, M.,Yamamoto, N., Yakaguaku Zasshi (Japan) 188, 2 1988), treatment ofEpsteinBarr virus (EBV) infections (Van Benschoten, M. M., Am. J.Acupunct, 16, 1 1988), and in the treatment of chronic hepatitis(Fujisawa, K. et al., Asian Med. J. (Japan), 23, 10 1980).

The anti-viral activity of GTPDs varies so unpredictably as to precludeany generalized statements as to whether such compounds have generalantiviral effect or even as to whether such compounds will generallyhave antiviral value as to any given virus. While GTPD drugs do, in someenvironments and under some conditions, exhibit some activity againstsome viruses, no anti-viral therapy based on GTPDs or in vitroanti-viral application of GTPDs has been generally accepted. TheAIDS-causing viruses, HIV-I and HIV-II, are the first retrovirusesidentified as pathogenic in man. While HIV are more fragile than mostinfectious viruses and are susceptible to destruction by mostvirus-inactivating methods, such as heating, use of glycerol compounds,etc., these methods also damage cells, e.g. the red blood cells, and,therefore, are not suitable for use in treating blood. In addition, anysubstance added to blood will, unless removed, remain in the blood, andmust, therefore, be non-toxic when administered intravenously. Removalof added toxins from blood is, at best, complex and expensive and maynot be feasible or possible without serious damage to blood components.It has now been discovered that glycyrrhizin, glycyrrhetinic acid,carbenoxolone and cicloxolone and the analogues thereof not onlyinactivate HIV in blood and are known to be well-tolerated intravenouslybut, in some instances, also serve as effective anticoagulants andcell-stabilizers and do not interfere with standard blood analyses.

The preferred method of carrying out the invention comprises providing atransfusion blood product container which contains an amount of the GTPDcompound, e.g. glycyrrhizin, glycyrrhetinic acid, carbenoxolone orcicloxolone to comprise from about 0.005 weight/percent (w/%) to about10 w/%, generally in the range of about 0.1 to about 3 w/% and a viralinactivator enhancer, e.g. glycerol or EDTA, or both,, of the collectedblood product when the container is filled, such amount being sufficientto inactivate CMV and/or other viruses in blood product collectedtherein from a donor, and substantially filling the container with thedonor's blood product. If the GTPD is to be used as the soleanticoagulant, a quantity sufficient to result in at least about 2 wt/%of the collected blood product should be in the collection container.The collected blood product is held for a sufficient period of time,e.g. 15 minutes or more at normal room temperatures or for an hour ormore at near 0° C., to assure that CMV is inactivated before the bloodproduct is administered by transfusion to the recipient patient. Carefuladmixing is essential and is preferably conducted immediately after thefirst mixing of blood product and GTPD's.

In carrying out this method, conventional blood product collectioncontainers are used. Such containers are typically made of sterilepolymer film and contain an anticoagulant. The collection, handling andadministration of the blood product by transfusion is the same as isconventionally carried out, save only for the processes involvingpreparation of the blood product to containing the GTPD compounds.

The GTPD compounds of this invention may be added to conventionalanticoagulants, e.g. citrate dextrose, citrate phosphate dextrose, EDTA,heparin, etc. to enhance the anticoagulant effect of these, or toreplace, in whole or in part, such anticoagulants.

There is no criticality respecting the addition of GTPD compounds at thetime of collection, as, for example, introducing blood product into bagscontaining GTPD compounds, though there are some advantages ofconvenience in this approach and this approach reduces the risk to bloodproduct handlers after collection. GTPD compounds may be added aftercollection any time in the chain of handling the blood product. It maybe desirable to assure a satisfactory inactivation of pathogenic virusin blood product to add GTPD compounds at two or more stages, such as atthe time of collection and 24 or 48 hours later, or at any later time.If the blood product is to be used for immediate transfusion, however,one careful thorough intermixing of a higher concentration of GTPDcompounds with a shorter delay between collection and transfusion thanwould normally occur in the routine handling of blood product.

The method of this invention is enhanced and the speed of virusinactivation is increased and the ultimate level of inactivation isimproved by including the step of maintaining the temperature of theblood product at from about 27° C. to about 60° C. if the blood productdoes not contain erythrocytes and to about 45° C. if the blood productcontains erythrocytes, or other heatsensitive components. The warming(or maintaining original warmth) ia carried on for at least about onehour, and as high as twenty four hours, or more, though longer heatingdoes not significantly improve the viral inactivation and is equivalentto up to twenty four hours insofar as the virus inactivation isconcerned. Temperatures of from about 37° C. to about 42° C. arepresently considered optimal for maximum enhancement of virusinactivation in blood products which contain erythrocytes or other heatsensitive constituents. Times as low as about one hour to twenty fourhours are considered optimum. There is an inverse relationship betweentime and temperature. Thus, a shorter time is required at about 45° C.than at 27° C. While longer times would be considered equivalent, as tovirus inactivation, it is considered unwise for other reasons, e.g.increased tendency toward hemolysis, to maintain the temperatures in theranges indicated for more than about 24 hours. A convenient time periodis about 12-16 hours, e.g. overnight and a temperature of about 37°C.±about 8° C. is considered optimum for blood products which containred blood cells. The blood product may be maintained at the indicatedtemperature soon after collection, just before transfusion, or at anyother time after the blood product and glycyrrhizic triterpenoid aremixed.

Illustrative, but not limiting, of one aspect of the invention, 1-MRC-5cells (Bartels) were grown in FCS and Eagle's minimal essential mediumwith 50 μg/ml of gentamicin, as the starting cell medium.2-Cytomegalovirus [ATCC;10⁷ tissue culture infectious dose (50)] wasadded to three samples each of (a) the media, (b) blood A and (c) bloodB. Glycyrrhizin in DMSO was added to one of the (a), (b) and (c) samplesto a final concentration of 2 wt/%. Controls containing only media addedto the same volume and DMSO in media were prepared. Six-hundred folddilutions were used to infect MRC-% monolayers grown in glass coverslipsinside glass vials. The inoculum was centrifuged at 1.000Xg for 1 hr. atroom temperature, and 1 ml of fresh media was added to each vial. Thecultures were incubated at 37° C. and observed daily for cytopathiceffect. Seven-days post-infection the monolayers were fixed withmethanol and stained by indirect fluorescent method using a CMVmonoclonal antibody (Syva). No cytopathic effect was observed in thesamples in which glycyrrhizin was present, while 3-4+cytopathic effectwas observed in the other samples. The fluorescent antibody techniqueshowed no evidence of residual virus in blood treated with GTPD. Bloodmixed with DMSO, and blood in media, tended to clot. Blood mixed withthe glycyrrhizic compound did not clot, but slight hemolysis may haveoccurred. pH adjustments using KOH or NaOH, etc., may be required.

Of the readily available GTPD compounds, carbenoxolone is preferred forits anti-viral effectiveness; however, carbenoxolone solutions are notstable for long periods of time and should be used freshly prepared. Inaddition, carbenoxolone lacks the powerful anti-coagulant effect which,surprisingly, is exhibited by glycyrrhizin and cicloxolone. Thus, withcarbenoxolone the user should include a proved anti-coagulant, theeffect of which is enhanced by the carbenoxolone.

It has been established with reasonable confidence that cicloxolone isless effective than carbenoxolone as an anti-viral agent in bloodproduct. On the other hand, however, cicloxolone and glycyrrhizinexhibit very surprising anti-coagulant effect, and, in addition arestable over relatively long periods of time in solution.

Having this discovery to build upon, it is apparent that an effectivemode of carrying out the present invention is to provide a mixture ofcarbenoxolone with either, or both, of glycyrrhizin or cicloxolone, thusobtaining both anti-viral anti-coagulant activity. If desired,anti-coagulant activity may be enhanced with other anti-coagulants, asmentioned above.

A most unusual phenomenon not suggest in any literature of which theinventor is aware was discovered which may be used to great advantage inhandling blood product supplies, and is useful in other facets of theinvention as well. When GTPDs are added to whole human blood productcontaining substantial amounts of lipids and lipoproteins the GTPD'sare, over a period of time, absorbed or adsorbed or otherwise removedfrom solution as active compounds. It is, accordingly, possible to treatblood product with an amount sufficient to inactivate the virus, e.g.CMV, in the blood product. Then, by delaying transfusion of the thustreated blood product for a sufficient period of time the blood productmay be transfused into the recipient free of such modest toxicity whichGTPD's in the concentration ranges under consideration may otherwiseintroduce into the blood product. The precise mechanism for this removalof GTPD compounds from the blood product solution has not beenelucidated with certainty; however, it is presently believed that theGTPD's are absorbed by the lipid or lipoprotein layers on red bloodcells or otherwise available in the blood. It is believed, also, that aparticular class of lipoproteins is either particularly active inremoving GTPD compounds from solution or, perhaps, are uniquely capableof such action. These postulated explanations are offered to aid inunderstanding and have not been firmly established, however, and theefficacy of the invention is not dependent upon the described action orupon any other postulated mode of action.

Another very significant discovery was made in the course of theinvestigation of GTPD compounds in blood. It was discovered that thepresence of GTPD's very significantly increased the clarity of plasmaand sera. A clear separation resulted from the plasma or serum and alayer believed to comprise lipids and/or lipoproteins which, in theabsence of GTPD's, gave the plasma or serum a cloudy, translucentappearance. Plasma and serum collected from blood to which GTPD's hadbeen added was crystal clear and transparent.

The GTPD compounds may be used in their acid form, as blood is a verypotent buffer; however, it is always necessary to check the pH afteradding the GTPD compound and, if necessary, adjust the pH to about7.0-8.0, e.g. with NaOH or KOH, before using the blood, as certain acidform GTPD compounds drop the pH of blood and plasma significantly to thepH 4-5 range.

The acid form of the GTPD compounds is only slightly soluble in waterbut is quite soluble in dimethyl sulfoxide. The salt, e.g. ammonium,sodium or potassium salts, of the GTPD compounds are, generally, solublein water, the sodium and potassium salts being more soluble than theammonium salts. It is, thus, convenient to purchase or prepare the GTPDcompounds as sodium or potassium salts.

One to the most surprising aspects of this invention is the discoverythat the addition of an appropriate concentration of GTPD compound(s) toblood tends to stabilize erythrocytes against lysing and other damage.

It has also been discovered that blood treated as described above, whenfractionated to produce plasma, clarifies the plasma, eliminating thetranslucence characteristic of most plasma.

Further, standard blood analyses, e.g. serum protein electrophoresis,basic blood chemistry tests, and lipid tests were unaffected by thepresence of the GTPD additives of this invention.

Thus, according to this invention, by a one-step addition of one or moreGTPD compounds to blood product at or after the time of collection (if asuitable anti-coagulant is used), the red blood cells are not only notlysed, but appear to be stabilized, CMV and other blood-borne viruses,e.g. HIV, are killed or inactivated, plasma is clarified, coagulation isinhibited, and conventional blood analysis are not significantlyeffected.

It has been established that the exemplary GTPD compounds glycyrrhizin,glycyrrhetinic acid, carbenoxolone and cicloxolone added to aconcentration of 1 w/% effectively reduces the CMV content by at leastone log, carbenoxolone being about 100 times as effective at the 1 w/%concentration as the other exemplary GTPD compounds. When theconcentration of GTPD compound is increased to 2 w/%, glycyrrhizin andcicloxolone each exhibited good anti-coagulant effect, sufficient topermit the omission of any other anticoagulant in most instances, whilethe limited anti-coagulant effect of carbenoxolone was insufficient. Inboth instances, however, a one, two or more log CMV inactivation wasachieved. In a comparable evaluation, a>3 log kill of HIV was achievedusing a 1 w/% carbenoxolone treatment.

A "blind" evaluation was carried out under the direction of the inventorby a scientist with wide experience in CMV infected blood. In thisevaluation, carbenoxolone was provided to the scientist withinstructions as to preparation of an aqueous solution thereof, butwithout disclosing the identity of the compound, carbenoxolone, to thescientist. The scientist determined that a 0.5% solution ofcarbenoxolone in whole blood achieved a 7 log kill, i.e. reduced CMV inthe blood by 7 logs (See Jakoby, W. H. and Pastan, I. H. (Eds), CELLCULTURE, (Volume LVIII of "Methods in Enzymology", Academic Press, Inc.,New York, Chapter 11, regarding measurement of cell inactivation) injust one-half hour and that 0.25% in blood gave a 2 log kill of CMV. Areciprocal relationship between time and concentration to achieveinactivation of animal fluid or tissue borne virus has now beenwell-established but only poorly quantified. A concentration of about0.1 percent present in blood for 12-24 hours is sufficient to achievesubstantially total kill or inactivation of CMV. If the blood contains ahigh EDTA, or both, in, lipid or lipoprotein level, however, it may bedesirable to treat the blood with two, three or four timed additions ofGTPD's to achieve a certainty of virus inactivation with lower overalladditons of GTPD compounds.

The effectiveness of GTPD compounds in killing or inactivating virus hasbeen verified in fetal bovine serum (FBS) where additions glycyrrhetinicacid in concentrations of 0.1 to 0.7 percent followed by adjustment topH 6.5 and 7.4, respectively for various trials, established a 100% killof the relatively resistant vesicular stomatitis virus (VSV) wasaccomplished in all cases.

The effectiveness of GTPD compounds as antiviral agents in blood is, inand of its self, a striking discovery. This discovery that GTPD added asdescribed stabilize red blood cells, and other blood cells such asplatelets, inhibit clotting, and stabilize proteins, such as FactorVIII, must be regarded as striking indeed.

The invention is embodied in an article of commerce comprising packagedtransfusion blood in a container of whole human blood containing one ormore glycyrrhizic triterpenoid compounds in an amount of from 0.005 to10 wt/%, preferably from about 0.1 to about 3 wt/% and a viralinactivator enhancer, e.g. glycerol or EDTA, or both, effective tosubstantially inactivate at least cytomegalovirus.

Glycerol and EDTA, most especially glycerol, in combination with GTPD asdescribed gives an unpredicted and unexpected synergism. Glycerol in lowconcentrations is not an effective anti-viral agent, though at very highconcentrations of glycerol, as is true of most hydroxy compounds, may insome instances inactivate some virus. Glycerol plus GTPD when present inconcentrations much lower than would provide anything akin to completeviral inactivation with glycyrol alone enhances the viral inactivationpower of the GTPD by at least one log and in some instances by threelogs or more. The optimum viral inactivator combination, as presentlyunderstood, comprises glycyrrhizic triterpenoid compounds in an amountof from 0.005 to 10 wt/%, preferably from about 0.1 to about 3 wt/% anda viral inactivator enhancer, e.g. fatty acid in a concentration of fromapproximately 0.0001 to 0.5 wt/% of the blood product mixture andpreferably a concentration of from approximately 0.0001 to 0.1 wt/% ofthe blood product mixture.

While other enhancers have not been studied to the extent that glycerolhas been, and specific data are still being accumulated, a synergisticamount of 3'-azido-3'-deoxythymidine, dextran, butyl hydroxy toluene,fatty acid or ethylene diamine tetraacetic acid and salts thereof, aswell as glycerol, in a concentration of from approximately 0.0001 to 0.5wt/% of the of the blood mixture, and preferrably in a concentration offrom approximately 0.0001 to 0.1 wt/% of the blood product mixture areconsidered, on the basis of available information and data, to beeffective.

The invention is also embodied in a container for collecting, treating,storing, handling or preparing whole human blood or transfusion bloodproduct for transfusion containing, as a part of the container, eitherin the main container or in a compartment of a multiple compartmentcontainer, one or more glycyrrhizic triterpenoid compounds in aconcentration of from 0.05 to 10.0 wt/%, preferably from about 0.5 toabout 3 wt/% and a viral inactivator enhancer, e.g. glycerol or EDTA, orboth, based on blood, sufficient to substantially inactivate virus in ablood product when the container is filled with such product.

Many blood containers have a large compartment for receiving the bloodproduct and one or more side pouches or side containers which have awall or a closure which, by appropriate physical manipulation, can beopened or ruptured to introduce the contents thereof into the largecompartment. Obviously, the nature of the container is immaterial solong as the end result, i.e. a container full of blood productcontaining the GTPD, can be accomplished with the container. Thus, theinvention is embodied in a transfusion blood container for theintroduction of one or more blood products, such as whole blood,platelet concentrations, leukocyte concentrations, plasma, plasmaderivatives, whole blood fractions, and combinations thereof, fortransfusing the patient, the invention comprising a transfusion bloodcontainer and an amount of one or more glycyrrhizic triterpenoidcompounds sufficient to comprise from 0.05 to 10.0 wt/%, preferably fromabout 0.5 to about 3 wt/% and a viral inactivator enhancer, e.g.glycerol or EDTA, or both,, of the contents of the container when fullof the blood product(s), sufficient to substantially inactivate virusescontained in the blood product introduced into said container.

As a method of collecting blood the invention is embodied in a processcomprising introducing said blood into a transfusion blood containercontaining glycyrrhizic triterpenoid compound sufficient to comprisefrom 0.05 to 10 wt/%, preferably from about 0.5 to about 3 wt/% and aviral inactivator enhancer, e.g. glycerol or EDTA, or both, of thecontents when the container is full.

The invention is also embodied in blood products resulting from thetreatment of blood with GTPD compounds. Such derivatives may include,for example, platelet and leukocyte concentrates, plasma, plasmaderivatives such as, for example, cryoprecipitate, panels of red bloodcells used in blood typing, and blood or blood fractions used for bloodanalysis such as, for example, the traditional blood samples nowroutinely collected in vacuum tubes. In such applications, the GTPDcompounds may be present in or added to the vacuum tubes or at any laterstage, though there are significant advantages in using vacuum tubescontaining GTPD compounds. Donor blood may be processed to yieldfollowing single-donor components. Multiple donor pools of plasmaharvested from whole blood can be processed to yield derivatives such asalbumin, plasma protein fraction, Factor VIII concentrate, immune serumglobulin preparation and concentrates of other blood factors. GTPDcompounds may with great advantage be added along with glycerol orglycerol-water before freezing blood factors or derivatives to obtain asynergistic stabilizing effect, namely the stabilizing of GTPD byglycerol and the stabilizing of the blood factor or derivative, or wholeblood in preparing cryoprecipitate, by both glycerol and the GTPDcompound(s). GTPD compounds may be used in cell wash solutions tostabilize blood cells, platelets and the like, and to prevent or inhibitcoagulation of the cells. It is advantageous, regardless of the mode orpurpose in processing blood, to inactivate pathogenic virus at theearliest reasonable stage in the handling chain and/or at specificpoints in the handling chain. The present invention is well adapted toany blood processing regime.

In all embodiments, the invention exhibits a number of surprisingresults. The spotty results reported in efforts to determine if, and towhat extent, GTPD compounds are indeed virucidal agents led the art tobelieve, as has been reported, that "the likelihood of developing ablood additive that would kill HIV and HBV and have no effect onlaboratory examination of blood seems small."(Peter C. Fuchs, M.L.O.,Oct. 1988, 13). In addition, notwithstanding the prior art in whichanti-viral activity, to the extent it exists, of GTPD compounds isuncertain, unpredictable and, as yet, unexplained, and the widelyaccepted proposition that no blood additive could be found which wouldinactivate blood-borne viruses without adversely effecting the blood,e.g. lysing the red blood cells and/or interfering with blood analyses,the present invention embodies processes and blood compositions in whichthese desired but hitherto unattainable results are accomplished.

Data indicate that carbenoxolone, over a comparatively short period oftime, about an hour or less, is bound by proteins and/or lipids and/orlipoproteins. Such data provide the basis for a nearly ideal method oftreatment of solutions and organs for transfusion or implantation is nowpossible. According to this nearly ideal method, carbenoxolone is addedto a fluid, such as blood, blood plasma, tissue culture medium ornutrient, or the like, which contains or to which protein, lipid, orlipoprotein is added, either contemporaneously or subsequently. Thevirus in the fluid are inactivated immediately, before the carbenoxoloneis completely bound, and, thereafter, the carbenoxolone is completelybound. If the protein, etc., is added, the addition can be effectedafter inactivation of the virus. When the fluid, e.g. blood or plasma,is transfused or the organ transplanted into the donee-patient, thefluid or organ is free of carbenoxolone. While carbenoxolone iswelltolerated, the ideal would be to avoid the introduction of anyforeign substance not necessary to the in vivo functioning of the fluidor implant organ. This ideal is attainable using the principles of thisinvention.

The GTPD compounds can be mixed with other active compounds withsynergistic results in inactivation of virus. Such synergistic andpotentially synergistic compounds include the anti-viral drug3'-azido-3'-deoxythymidine (AZT), which acts synergistically with theGTPD compounds, dextrans, butyl hydroxy toluene, fatty acids such asoleic acid, chelating agents such as EDTA, and compounds of transitionand heavy metals. Highly beneficial effects with minor, if any, adverseside effects have been accomplished using a glycerol, or ethylenediamine tetraacetic acid and salts thereof (EDTA) in very lowconcentrations of from approximately 0.0001 to 5 wt/%, preferably 0.0001to 0.01 wt/%.

In the case of blood, plasma or other fluid collection in a bag, vacuumtube, vial or other container, a highly desirable and preferred methodand apparatus are utilized. The GTPD compound, in dry form, is in orassociated with the container and is dissolved and added to the fluideither immediately before collection or soon afterward. Since some formsof the GTPD compounds decompose in solution, a fresh source of the GTPDcompounds is provided by the method just described.

INDUSTRIAL APPLICATION

This invention has direct application in the blood banking industry.

What is claimed:
 1. Transfusion blood product comprising a transfusioncontainer containing human blood product and one or more glycyrrhizictriterpenoid compounds in an amount of from 0.005 to 10 wt/% and asynergistic amount of 3'-azido-3'-deoxythymidine, dextran, butyl hydroxytoluene, fatty acid, glycerol or ethylene diamine tetraacetic acid andsalts thereof of concentrations of from approximately 0.0001 to 5 wt/%effective to substantially inactivate susceptible viruses found inanimal fluids and tissues.
 2. The transfusion blood product of claim 1comprising from about 0.1 to about 3 wt/% glycyrrhizic triterpenoidcompounds.
 3. The transfusion blood product of claim 2 wherein at leastone glycyrrhizic triterpenoid compound is carbenoxolone.
 4. Thetransfusion blood product of claim 2 wherein at least one glycyrrhizictriterpenoid compound is cicloxolone.
 5. The transfusion blood productof claim 4 wherein the synergistic amount of 3'-azido-3'-deoxythymidine,dextran, butyl hydroxy toluene, fatty acid, glycerol or ethylene diaminetetraacetic acid and salts is present in a concentration of fromapproximately 0.0001 to 0.1 wt/% of the blood product when the containeris full.
 6. A method for preparing human blood product for transfusion,comprising mixing such blood product with one or more glycyrrhizictriterpenoid compounds in a concentration of from 0.05 to 10.0 wt/% anda synergistic amount of 3'-azido-3'-deoxythymidine, dextran, butylhydroxy toluene, fatty acid, glycerol or ethylene diamine tetraaceticacid and salts thereof of concentrations of from approximately 0.0001 to5 wt/% based on blood product and holding the resulting mixture for aperiod of at least about one hour, the concentration being sufficient tosubstantially inactivate susceptible viruses found in animal fluids andtissues within said time.
 7. The method of claim 6 comprising mixingsaid glycyrrhizic triterpenoid compounds into such blood product in aconcentration of from about 0.1 to about 3 wt/%.
 8. The method of claim7 comprising the mixing of the synergistic amount of3'-azido-3'-deoxythymidine, dextran, butyl hydroxy toluene, fatty acid,glycerol or ethylene diamine tetraacetic acid and salts into such bloodproduct in a concentration of from approximately 0.0001 to 0.1 wt/%. 9.The method of claim 8 wherein the holding time is at least about 12hours.
 10. The method of claim 8 further comprising adding an additionalamount of said glycyrrhizic triterpenoid compounds into such bloodproduct approximately at or after the end of the first holding period torenew the concentration thereof to from about 0.1 to about 3 wt/% andholding the resulting mixture for a second holding period of at leastabout 12 hours.
 11. The method of claim 7 further comprising adding anadditional amount of said glycyrrhizic triterpenoid compounds into suchblood product approximately at or after the end of the first holdingperiod to renew the concentration thereof to from about 0.1 to about 3wt/% and holding the resulting mixture for a second holding period of atleast the same duration.
 12. The method of claim 7 further comprisingthe step of holding the mixture for a further holding time of at leastabout 12 hours sufficient to permit substantially all of theglycyrrhizic triterpenoid compounds to be removed from solution forsubstantially eliminating any possible toxicity of the glycyrrhizictriterpenoid compounds to the recipient of the blood product.
 13. Themethod of treating a patient comprising transfusing the patient withblood product comprising one or more glycyrrhizic triterpenoid compoundsin a concentration of from 0.005 to 10.0 wt/% and a synergistic amountof 3'-azido-3'-deoxythymidine, dextran, butyl hydroxy toluene, fattyacid, glycerol or ethylene diamine tetraacetic acid and salts thereof ina concentration of from approximately 0.0001 to 5 wt/% based on bloodproduct sufficient to substantially inactivate susceptible viruses foundin animal fluids and tissues.
 14. The method of claim 13 wherein thesynergistic amount of 3'-azido-3'-deoxythymidine, dextran, butyl hydroxytoluene, fatty acid, glycerol or ethylene diamine tetraacetic acid andsalts is present in a concentration of from approximately 0.0001 to 0.1wt/%.
 15. The method of claim 14 wherein said blood product comprisesglycyrrhizic triterpenoid compounds in a concentration of from about 0.1to about 3 wt/%.
 16. The method of claim 14 wherein the blood producthas been retained for a total holding time after being mixed with saidcompounds of at least about 12 hours sufficient to permit substantiallyall of said glycyrrhizic triterpenoid compounds to be removed fromsolution for substantially eliminating any possible toxicity of theglycyrrhizic triterpenoid compounds to the recipient patient.
 17. Themethod of collecting blood product comprising introducing said bloodproduct into a transfusion blood product container containingglycyrrhizic triterpenoid compound sufficient to comprise from 0.005 to10 wt/% and a synergistic amount of 3'-azido-3'-deoxythymidine, dextran,butyl hydroxy toluene, fatty acid, glycerol or ethylene diaminetetraacetic acid and salts present in a concentration of fromapproximately 0.0001 to 0.5 wt/% of the contents when the container isfull and mixing the contents of the container.
 18. The method of claim17 wherein the synergistic amount of 3'-azido-3'-deoxythymidine,dextran, butyl hydroxy toluene, fatty acid, glycerol or ethylene diaminetetraacetic acid and salts is present in a concentration of fromapproximately 0.0001 to 0.1 wt/% of the contents when the container isfull.
 19. The method of collecting blood product of claim 17 wherein theamount of glycyrrhizic triterpenoid compound is sufficient to comprisefrom about 0.1 to about 3 wt/% of the contents when the container isfull.
 20. The method of claim 19 wherein the synergistic amount of3'-azido-3'-deoxythymidine, dextran, butyl hydroxy toluene, fatty acid,glycerol or ethylene diamine tetraacetic acid and salts is present in aconcentration of from approximately 0.0001 to 0.1 wt/% of the contentswhen the container is full.
 21. A method for preparing blood product forprocessing to recover blood constituents, fractions and components,comprising mixing such blood product with one or more glycyrrhizictriterpenoid compounds in a concentration of from 0.05 to 10.0 wt/% anda synergistic amount of 3'-azido-3'-deoxythymidine, dextran, butylhydroxy toluene, fatty acid, glycerol or ethylene diamine tetraaceticacid and salts is present in a concentration of from approximately0.0001 to 5 wt/% based on blood product and holding the resultingmixture for a period of at least about one hour, the concentration beingsufficient to substantially inactivate susceptible viruses found inanimal fluids and tissues within said time and then further processingthe blood product to recover blood constituents.
 22. The method of claim21 comprising mixing said glycyrrhizic triterpenoid compounds into suchblood product in a concentration of from about 0.1 to about 3 wt/%. 23.The method of claim 22 wherein the holding time is at least about 12hours.
 24. The method of claim 23 further comprising adding anadditional amount of said glycyrrhizic triterpenoid compounds into suchblood product approximately at or after the end of the first holdingperiod to renew the concentration thereof, the amount being from about0.1 to about 3 wt/% and holding the resulting mixture for a secondholding period of at least about 12 hours.
 25. The method of claim 24further comprising adding an additional amount of said glycyrrhizictriterpenoid compounds into such blood product approximately at or afterthe end of the first holding period to renew the concentration thereof,the amount being from about 0.1 to about 3 wt/% and holding theresulting mixture for a second holding period of at least the sameduration.
 26. The method of claim 21 wherein the synergistic amount of3'-azido-3'-deoxythymidine, dextran, butyl hydroxy toluene, fatty acid,glycerol or ethylene diamine tetraacetic acid and salts is present in aconcentration of from approximately 0.0001 to 0.1 wt/%.
 27. The methodof claim 26 comprising mixing said glycyrrhizic triterpenoid compoundsinto such blood product in a concentration of from about 0.1 to about 3wt/%.
 28. The method of claim 27 wherein the holding time is at leastabout 12 hours.
 29. The method of claim 27 further comprising adding anadditional amount of said glycyrrhizic triterpenoid compounds into suchblood product approximately at or after the end of the first holdingperiod to renew the concentration thereof to from about 0.1 to about 3wt/% and holding the resulting mixture for a second holding period of atleast the same duration.
 30. The method of any of claims 6, 17, or 21further comprising the step of maintaining the temperature of the bloodproduct at from about 37° C.±about 8° C. for from about one to twentyfour hours after the blood product and the glycyrrhizic triterpenoidcompound are mixed.
 31. The method of any of claims 6, 17, or 21 whereinthe blood product does not contain erythrocytes or other heat sensitiveconstituents and the process further comprises the step of maintainingthe temperature of the blood product at from about 27° C. to about 60°C. for form about one to twenty four hours after the blood product andthe glycyrrhizic triterpenoid compound are mixed.